Vascular smooth muscle cell (VSMC) proliferation and migration are essential for vascular development, angiogenesis and repair; their dysfunction contributes to vascular diseases such as atherosclerosis, hypertensive microvessel remodeling and Leiomyosarcomas. In these diseases VSMC switch from a quiescent differentiated and contractile phenotype to a synthetic proliferative and migratory phenotype, a condition that can be recapitulated in culture. There is clear evidence that this VSMC phenotypic modulation is of paramount clinical importance in atherosclerosis and other vascular occlusive diseases, yet the molecular mechanisms of this modulation remain incompletely understood. VSMC phenotypic modulation is accompanied by a change in ion channel expression: synthetic VSMC downregulate the expression of L-type Ca2+ channels and upregulate that of canonical transient receptor potential (TRPC) channels. Our preliminary studies have demonstrated an increase in TRPC6 and the newly discovered calcium sensor STIM1, and calcium channels Orai1 and Orai3 expression in synthetic cultured rat aortic VSMC, as compared to quiescent freshly isolated cells. We hypothesize that STIM1 is a master regulator of Ca2+ signaling in VSMC required for Orai1, Orai3 and TRPC6 channel function and that increased Ca2+ entry as a result of STIM1, Orai and TRPC upregulation contribute to VSMC proliferation and migration in disease. In support of this hypothesis we found that STIM1 knockdown using silencing RNA (siRNA) inhibited VSMC proliferation in culture and we revealed agonist-specific activation of distinct Ca2+ channels and remarkable STIM1 versatility in regulating these channels. Indeed, knockdown of STIM1 in synthetic VSMC abrogated the function of: i) PDGF-activated Orai1 channels; ii) thrombin-activated channels contributed by heteromultimeric Orai1/3 and iii) Diacylglycerol (DAG)-activated TRPC6 channels. In Aim1 we will biophysically characterize thrombin-activated Ca2+ entry pathways using whole cell patch clamp and determine the role of STIM1 oligomerization, cellular localization and interaction with Orai1 and Orai3 in the activation of this Ca2+ entry pathway. In Aim2 we will determine whether sarcoplasmic reticulum- or plasma membrane- associated STIM1 is involved in TRPC6 activation by DAG, examine the role of STIM1 oligomerization and the interaction of STIM1 and TRPC6 by FRET microscopy and the interaction of their native counterparts using co-immunoprecipitations. In Aim3 we will test the hypothesis that Orai1, Orai3 and TRPC6 upregulation also occurs in vivo in a rat model of vascular injury and determine the effect on neointimal formation of in vivo silencing of these proteins using adenovirus encoding siRNA. The results from this proposal will generate a better understanding of VSMC physiology and unveil novel targets for drug therapy aimed at controlling VSMC proliferation and migration that occur during vascular diseases such as atherosclerosis and hypertension. PUBLIC HEALTH RELEVANCE: The results from this proposal will generate a better understanding of VSMC physiology and unveil novel targets for drug therapy aimed at controlling VSMC proliferation and migration that occur during vascular diseases such as atherosclerosis and hypertension.